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研究生:林冠佑
研究生(外文):Guan-You Lin
論文名稱:沙門氏菌藉由調控C-X-C趨化因子受體-4降低腫瘤的轉移
論文名稱(外文):Salmonella reduces tumor metastasis by downregulation C-X-C chemokine receptor type 4
指導教授:李哲欣李哲欣引用關係
指導教授(外文):Lee, Che-Hsin
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:61
中文關鍵詞:CXCL12CXCR4腫瘤轉移沙門氏菌細菌療法
外文關鍵詞:Bacterial therapyCXCL12CXCR4MetastasisMigrationSalmonella
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腫瘤轉移是癌症致死率高的原因,研究指出C-X-C chemokine receptor type 4 (CXCR4) 會在多種癌細胞中過度表達,並只會與其專一配體C-X-C chemokine ligand 12 (CXCL12) 結合,促使血管新生、腫瘤轉移。常見的癌症療法已受到侷限,以細菌治療腫瘤成為新趨勢,沙門氏菌是一種兼性厭氧的革蘭氏陰性菌,已被證實有抗腫瘤的活性。我們透過西方墨點法、細胞遷移試驗 (wound healing as-say及transwell migration assay) 證實沙門氏菌在小鼠黑色素瘤及小鼠肺癌細胞中能有效抑制CXCR4蛋白質表現並同時降低腫瘤細胞的移動能力。為了確定沙門氏菌是透過Protein Kinase-B (Akt)/Mammalian Target of Rapamycin (mTOR) 路徑來調控CXCR4,我們轉染持續活化的Akt基因進入細胞,結果顯示Akt的增加會減弱沙門氏菌對CXCR4蛋白質的抑制及細胞移動能力的抑制。我們在transwell migration assay的下室中添加CXCL12,驅使更多的癌細胞移動到下室,但是沙門氏菌會抑制此現象。在動物實驗中,我們將預先處理及未處理過沙門氏菌的腫瘤細胞透過尾靜脈注射到小鼠體內循環,大約18天 後從小鼠體內取出肺部,觀察外觀及秤重後發現控制組可以看到明顯的腫瘤,在重量上也大於沙門氏菌處理過的組別。從以上細胞實驗及動物實驗結果證實CXCL12會驅使腫瘤細胞移動,而沙門氏菌能有效抑制CXCR4表達及腫瘤細胞的移動,並抑制小鼠體內的腫瘤轉移。
Tumor metastasis is the main reason for the death of most cancer patients. C-X-C chemokine receptor type 4 (CXCR4) has been proven to be overexpressed in numerous types of cancer. CXCR4 selectively binds with C-X-C chemokine ligand 12 (CXCL12), which improved tumor proliferation and metastasis. Recently, some limitations are reported on the use of conventional cancer treatments; bacteria treatment for cancer becomes a trend that overcomes these limitations. Salmonella is a gram-negative fac-ultatively anaerobe, plenty of studies show that Salmonella has antitumor activity. In this study, Salmonella suppresses CXCR4 protein expression and tumor cell migration ability in B16F10 melanoma and LL2 lung carcinoma cells. We show here that Salmo-nella suppresses CXCR4 through downregulating Protein Kinase-B (Akt)/Mammalian Target of Rapamycin (mTOR) signaling pathway. In cells transfected with constitu-tively active myr-Akt plasmid, we observed a reverse effect of Salmonella-induced inhibition of CXCR4. We confirmed that tumor cells have chemotactic response to CXCL12 in transwell migration assay, and we found that Salmonella diminishes this response. The C57BL/6 mice were intravenously injected with B16F10 and LL2 cells pre-incubated with or without Salmonella. At day 18, the tumor size and lung weight of Salmonella group had obviously decreased, indicating anti-metastatic effect that con-firmed the findings from the in vitro experiments.
 目錄
論文審定書 i
摘要 ii
Abstract iii
目錄 iv
圖次 vi
縮寫 vii
第一章、緒論 1
1. 轉移 (metastasis) 1
2. 趨化因子及趨化因子受體 (chemokine and chemokine receptor) 2
3. C-X-C 趨化因子受體-4 (C-X-C Chemokine receptor type-4) 4
4. C-X-C趨化因子配體-12 (C-X-C Chemokine motif ligand 12) 5
5. 細菌療法 (Bacterial therapy) 6
6. 沙門氏桿菌 (Salmonella) 7
第二章、研究動機與目的 9
第三章、材料與方法 10
1. 材料 (Material) 10
1.1 細胞株 (Cell line) 10
1.2 抗體 (Antibodies, Ab) 10
1.3 質體 (Plasmid) 11
1.4 試劑 (Reagents) 11
1.5 實驗器材 (Equipments) 14
1.6 儀器 (Instruments) 15
1.7 緩衝液 (Buffer) 18
2. 方法 (Method) 19
2.1 細胞解凍 (Cell thawing) 19
2.2 細胞繼代 (Cell subculture) 20
2.3 細胞冷凍保存 (Cryopreservation) 20
2.4 細胞計數 (Cell counting) 21
2.5 沙門氏桿菌培養 (Salmonella culture) 22
2.6 使用沙門氏菌感染細胞 (Cell infections by Salmonella) 22
2.7 轉染 (Transfection) 23
2.8 傷口癒合試驗 (Wound healing assay) 24
2.9 Transwell migration assay 24
2.10 轉移性試驗 (Metastatic experiment) 24
2.11 西方墨點法 (Western blotting) 25
2.12 動物實驗 (Animal study) 28
2.13 統計分析 (Statistical analysis) 28
第四章、研究結果 29
1. 沙門氏菌對於B16F10及LL2細胞遷移能力的影響 29
2. 沙門氏菌透過Akt/mTOR路徑調控CXCR4表現量 30
3. 沙門氏菌對於持續活化Akt細胞蛋白質表達的影響 30
4. 藉由wound healing assay觀察沙門氏菌對於持續活化Akt細胞遷移能力的影響 31
5. 沙門氏菌對於CXCL12吸引CXCR4促進細胞遷移的影響 32
6. 沙門氏菌對於小鼠體內腫瘤轉移的影響 32
第五章、討論 34
第六章、結論 38
參考文獻 39
圖表 45


圖次
Figure 1. Influences of Salmonella on tumor metastasis ability. 45
Figure 2. Effects of Salmonella on protein expression in B16F10 and LL2 cell lines. 46
Figure 3. Constitutively active Akt reduced the effects of Salmonella on CXCR4 expression. 47
Figure 4. Constitutively active Akt reduced the influences of Salmonella on cell migration. 48
Figure 5. Tumor cells have positive chemotaxis toward CXCL12. 49
Figure 6. Salmonella decreased metastasis of tumor cells in vivo mouse model. 50
Figure 7. The roles of CXCR4/CXCL12 in cancer cells 51
Figure 8. Illustration of salmonella downregulation CXCR4. 52
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